Fig. 4: Ym1 directly interacted with WYMV CP to trigger cell death.

A Diagrams showing the constructs of full-length Ym1 and its truncated versions. The truncated Ym1 domains were fused to the C-terminal of the Green fluorescent protein (GFP) tag and expressed in N. benthamiana leaves by Agrobacterium-mediated transient transformation. Grey: complete Ym1 protein; Yellow: CC domain; Red: NBS domain; Blue: LRR domain. The numbers indicate the starting or stopping amino acid (aa) sites of the tested domains. B Y2H assays revealing the critical domains for WYMV CP-Ym1 interaction. The corresponding sequences were fused to the Gal4 DNA-binding domain (BD); the WYMV CP was fused to the Gal4 activation domain (AD). The yeast co-transformed with BD and AD constructs were plated onto synthetic dropout (SD) media lacking Leu and Trp (left) and SD media lacking Ade, His, Leu, and Trp (right). Images were taken at 3 days after incubation. C Co-immunoprecipitation (Co-IP) analysis showing the interactions between Ym1-GFP fusion protein or its truncated domain LRR-GFP fusion protein with WYMV CP in N. benthamiana leaves. Total proteins and IP products were detected by Western blotting using anti-HA or anti-GFP antibodies at 48 hpi. The arrows point the bands for Ym1-GFP (upper) and LRR-GFP (lower). The experiment was repeated three times independently with similar results. D LUC interaction of Ym1 or its truncated LRR domain with WYMV CP detected by split luciferase complementation imaging (LCI) assay on N. benthamiana leaves. The leaf areas infiltrated with Agrobacterium cultures expressing nluc-CP+cluc, nluc+cluc-Ym1, and nluc+cluc-LRR were used as negative controls, respectively. Luciferase activity was captured using an imaging apparatus at 2 days post inoculation. E Cell death triggering activity assay of Ym1 and its truncated domains or domain combinations. Each image was taken at 96 h post-inoculation (hpi). F Phenotypic observations of Ym1-mediated WYMV resistance elicited by WYMV CP. Triggered hypersensitive response (HR) cell death was observed in N. benthamiana leaves when co-expressing Ym1-GFP and CP-HA combinations, but not observed when co-expressing the remaining tested combinations. Fusion proteins were expressed in N. benthamiana leaves by Agro-infiltration. The trypan blue staining was conducted to validate the cell death result. G Cell death assay of different genotypes in wheat roots after artificial inoculation of WYMV infectious clones. Bars = 50 μm. The experiment was repeated three times independently with similar results.